Cytoplasmic heat shock protein 90 alpha and beta (Hsp90? and ?) and endoplasmic reticulum (ER) glucose-regulated protein94 (Grp94) are the main known mammalian Hsp90 paralogs. Each is responsible for chaperoning a distinct set of client proteins and has a unique biochemical role, despite 50% sequence identity in N-domains and analogous regulatory ligand binding cavities. While Hsp90? and ? have important roles in maintaining the functional conformation of a large number of aberrant malignancy- and neurodegeneration- driving proteins, such as kinases, transcription factors and anti-apoptotic proteins, Grp94 lacks these biochemical hallmarks. In contrast, Grp94 is involved in the regulation of a restricted number of proteins involved in channeling immune and inflammatory signals. Of especial importance are emerging therapeutic implications of Grp94 in regulating the immune response in many pathological conditions, in which this mainly ER chaperone, translocates to the cell surface and/or is excreted into the circulation. Through regulation of cell surface expression of Toll-like receptors (TLRs), Grp94 is associated with pathological processes like autoimmune disease, chronic inflammatory conditions and sepsis. Recently, a link between Grp94 and the pathogenesis of autoimmune diabetes and the development of vascular complications, frequently associated with the disease, has emerged. Furthermore, Grp94 is implicated in promoting chronic inflammation in rheumatoid arthritis. Taken together, these findings suggest Grp94 as a valid target for therapeutic intervention, and position the Grp94 inhibitors as potential therapeutics in the treatment of immune-related disorders. To date however, no selective Grp94 inhibitor scaffold of therapeutic significance has been reported, and all inhibitors in clinical evaluation for cancers act with similar affinity on all four Hsp90 members. The use of pan-Hsp90 inhibitors for immune-related disorders, of which most require chronic administration, has its obvious limitations, and may be associated with unacceptable benefit to toxicity ratio. To overcome these limitations, this application proposes to identify selective and therapeutically relevant Grp94-small molecule inhibitor scaffolds. While challenging because of the high structural similarity among Hsp90s in the ligand binding pocket, our preliminary data confirm that discovery of Grp94 inhibitors with several log-orders of selectivity over Hsp90s is possible. Our proposal offers a pioneering perspective on the discovery, design, synthesis and evaluation of selective Grp94 inhibitors. It will employ an iterative feed-back strategy in which computational, synthetic and medicinal chemistry efforts feed into and from biochemical and biological information. At the end of the two years we anticipate to have gained important SAR information that will teach on the structural features imperative for a lead Grp94 inhibitor. Our ultimate goal is to identify leads for future clinical translation. PUBLIC HEALTH RELEVANCE: The molecular chaperone Grp94, unlike is closely related paralog Hsp90, is involved in the regulation of a restricted number of proteins involved in channeling immune and inflammatory signals, indicating that the pathogenic Grp94 roles are amenable for selective therapeutic modulation. As such, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis and stroke can be potentially treated by targeting Grp94 with small molecule inhibitors. Selective small molecules of the Grp94 chaperone with therapeutic applicability are yet to be reported, and our application is first to describe strategies for the discovery and development of such agents.